Figure 3
ORF2 protein interferes with IκBα ubiquitination. (A) Huh7 cells transfected with pSGI vector, ORF2 (full-length) and Δ35 ORF2 expression constructs were labeled metabolically with [35S]-cys/met promix and treated with MG-132 for 2 hours prior to lysis. Lysates were immunoprecipitated with anti-IκBα antibody and immunoblotted with anti-ubiquitin antibody (upper panel). Same blot was stripped and reprobed with IκBα antibody (middle panel). An aliquot of the lysate was immunoprecipitated with anti-ORF2 antibody and radioactive bands were detected by autoradiography (lower panel). (B) Huh7 cells transfected with pSGI (lane 1) or ORF2 (increasing DNA amounts, lane 2 and 3) expression plasmids were metabolically labeled with [35S]-cys/met promix. Lysates were immunoprecipitated with IκBα antibody and immunoblotted with SKP1, CUL1 and IκBα antibodies. An aliquot of lysate was immunoprecipitated with anti-ORF2 antibody and radioactive bands were detected by autoradiography. Graph represents percent band intensities of Skp1, Cul1 and IκBα (from lanes 1–3) levels, considering the intensity of pSGI transfected band in lane 1 as 100%. Similar results have been obtained in 3 independent experiments. (C) Huh7 cells expressing the indicated protein were immunoprecipitated with the respective antibodies and immunoblotted anti-IκBα antibody (upper panel). Same blot was stripped, cut into halves and immunoblotted with anti-myc (lower pane, lane 1–5) or anti-ORF2 (lane 6–8) antibodies. In lane 1, 50% of the sample has been loaded in comparison to other lanes. Graph represents percent IκBα band intensity, considering the intensity of IκBα band in lane 2 as 100%. Similar results have been obtained in 3 independent experiments. (D) pSGI vector (lane 3) or pSGI ORF2 (lane 1 and 2) transfected cells were labeled with [35S]-cys/met promix and lysates were immunoprecipitated with anti-IκBα antibody, pre-immune serum (PS) or anti-ORF2 antibodies, respectively; subsequently, samples were immunoblotted with SKP1 and CUL1 antibodies. Same blot was air dried and exposed to X-ray film to check for ORF2 expression by autoradiography.